Epithelial tissues comprise a layer or layers of cells that cover free and enclosed surfaces throughout the body, including cutaneous, mucous, lumenal, serous, and glandular spaces. All epithelial layers contain two specialized domains: an apical domain that faces the mucosal (or lumenal) space and a basolateral membrane that faces the serosal (or ablumenal) space. Thus an important function of all epithelia is to provide an appropriate barrier function to separate and to control many physiological processes between these two spaces. In the lung, for example, the airways epithelia serve many functions, including providing a barrier between the lung mucosa and blood supply, to coordinate the hydration of the airways, to regulate blood-borne immune responses in the airway mucosa, and to clear the airways of toxins and pathogens. Epithelial cells are ubiquitous throughout the body, and are found in the entire respiratory and digestive tract, reproductive system and sensory organs (eye, ear, nose and skin). Epithelial cells have evolved to serve many homeostatic functions that are specific to their location throughout the body. One such specific function is found in the mucociliary clearance (MCC) system. Mucous secretions are normally removed via the MCC. MCC relies on the integrated action of three components: 1) mucus secretion by goblet cells and submucosal glands; 2) the movement of cilia on epithelial cells which propels the mucus across the luminal surface; and 3) ion transport into and out of luminal epithelial cells which concomitantly controls the flow of water into the mucus. It is now known that nucleoside phosphates such as uridine 5′-triphosphate (UTP) modulate all of the components of the MCC system. First, UTP has been shown to increase both the rate and total amount of mucin secretion by goblet cells in vitro (M. Lethem, et al., Am J. Respir. Cell Mol. Biol. 9, 315-22 (1993)). Second, UTP has been shown to increase cilia beat frequency in human airway epithelial cells in vitro (D. Drutz, et al., Drug Dev. Res. 37(3), 185 (1996)). And third, UTP has been shown to increase Cl− secretion, and hence, water secretion from airway epithelial cells in vitro (S. Mason, et al., Br. J. Pharmacol. 103, 1649-56 (1991)). In addition, it is thought that the release of surfactant from Type II alveolar cells in response to UTP (Gobran, Am. J. Physiol. 267, L625-L633 (1994)) contributes to optimal functioning of the lungs and may assist in maximizing MCC. UTP has been shown to increase intracellular Ca++ due to stimulation of phospholipase C by the P2Y2 receptor (H. Brown, et al., Mol. Pharmocol. 40, 648-55 (1991)).
The retinal pigment epithelium (RPE) lies in the back of the vertebrate eye and forms a barrier that separates the retina from the choroidal blood supply. Although anatomically an epithelial tissue, the RPE also functions in a glial-like capacity in maintaining homeostatic retinal function. For example, a critical function of the RPE is to maintain and regulate the hydration of the subretinal space, the extracellular volume that exists between the retina and the RPE. (Marmor, pp. 3-12, in The Retinal Pigment Epithelium, Eds. M. F. Marmor and T. J. Wolfensberger, Oxford University Press, New York, (1998)) This function is achieved by the regulated transport of fluid, ions, and metabolites between the subretinal space and the choroidal blood supply. (Marmor, pp. 420-438, in The Retinal Pigment Epithelium, Eds. M. F. Marmor and T. J. Wolfensberger, Oxford University Press, New York, (1998); Pederson, pp. 1955-1968, in Retina, Ed. S. J. Ryan, Mosby, St. Louis, (1994)). Like all epithelia, the RPE contains two functionally and anatomically distinct membranes: an apical membrane that faces the retina, and a basolateral membrane that faces the choroidal blood supply. In the normal retina, fluid is absorbed across the RPE in the direction of the subretinal space to the choroid. This active absorption of fluid by the RPE, often referred to as the “RPE pump,” plays a critical role in maintaining proper attachment of photoreceptors to the apical membrane of the RPE by pumping fluid out of the retinal spaces. (Marmor, pp. 1931-1954, in Retina, Ed. S. J. Ryan, Mosby, St. Louis, (1994); Hughes, et al., pp. xvii, 745, in The Retinal Pigment Epithelium, Eds. M. F. Marmor and T. J. Wolfensberger, Oxford University Press, New York, (1998)).
Glaucoma is a disease complex characterized primarily by an increase in intraocular pressure. Sufficiently high and persistent intraocular pressure may result in damage to the optic disc at the juncture of the optic nerve and retina, resulting in irreversible blindness. There are three types of glaucoma: primary, secondary, and congenital. Primary glaucoma is subdivided into narrow angle (acute congestive) and wide-angle (chronic simple) types, depending on the configuration of the angle of the anterior chamber where re-absorption of the aqueous humor occurs. Effects on the volumes of the various intraocular vascular beds, such as those of the iris and ciliary body and on the rate of secretion of the aqueous humor into the posterior chamber may contribute secondarily to the lowering of the pressure or, conversely, may produce a rise in pressure preceding the fall. In narrow angle glaucoma, the aqueous outflow is enhanced by freeing of the entrance to the trabecular space at the canal of Schlemm from blockade by the iris, as a result of the drug-induced contraction of the sphincter muscle of the iris. (Taylor, pp.123-125, in The Pharmacological Basis of Therapeutics, 7th Ed, Eds., A. G. Gilman, L. S. Goodman, T. W. Rall, and F. Murad, MacMillan Publishing Company, New York, (1985))
In wide-angle, or chronic simple, glaucoma, the entry to the trabeculae is not physically obstructed; the trabeculae, a meshwork of pores of small diameter, lose their patency. Contraction of the sphincter muscle of the iris and the ciliary muscle enhances tone and alignment of the trabecular network to improve re-absorption and outflow of aqueous humor through the network to the canal of Schlemm (Watson, Br. J. Opthalmol. 56: 145-318 (1972); Schwartz, N. Engl. J. Med., 290: 182-186 (1978); Kaufmnan, et al., Handbook of Experimental Pharmacology 69: 149-192 (1984)).
Human joints are lubricated by fluid secreted from synovial membranes, which line internal, non-articular joint surfaces. The lubricating properties of synovial fluid have been attributed to a surfactant consisting of surface active phospholipid (SAPL), the mucinous glycoprotein lubricin, hyaluronic acid (hyaluronan), and water. Hyaluronan is a critical constituent component of normal synovial fluid and an important contributor to joint homeostasis. Hyaluronan imparts anti-inflammatory and antinociceptive properties to normal synovial fluid and contributes to joint lubrication, buffering load transmission across articular surfaces and providing a continually replenished source of hyaluronan to articular tissues. Joint lubrication is compromised in osteoarthritis (OA).
Studies suggest that activation of P2Y receptors by extracellular nucleotides elicit responses from inflammatory cells (such as mast cells, eosinophil, leukocytes, neutrophils) consistent with a pro-inflammatory effect. Extracellular nucleotide-induced stimulation of leukocytes and subsequent adhesion to endothelium has been shown to play an important role in inflammatory diseases. Extracellular nucleotides stimulate P2Y receptor on human polymorphonuclear neutrophils (PMN) with the pharmacological profile of the P2Y2 receptor.
Allergy is a state of hypersensitivity caused by exposure to a specific antigen (allergen) resulting in harmful immunologic reactions or subsequent exposures. The first encounter with an allergen sensitizes the body via the lymphocytes, resulting in IgE coating of mast cells and basophils. Subsequent exposure results in the development of the “early phase” of the allergic reaction and occurs within seconds or minutes of exposure to an allergen. The early phase is also known as the immediate hypersensitivity reaction. In the allergic reaction, hypersensitivity is a condition in a previously exposed person, in which tissue inflammation is caused by an immune reaction upon re-exposure to an allergen sensitizer. In half of occurrences, the allergic reaction develops into a “late phase,” which occurs about 4 to 6 hours after the exposure. In the late phase reaction, tissues become red and swollen due to the collection of eosinophils, neutrophils, lymphocytes, and other cells.
Previous work has demonstrated the presence of P2Y receptors in glial and neuronal cells of the mature nervous system (Abbracchio and Bumstock, Jpn J. Pharmacol, 78:113-45, 1998). P2Y receptors belong to a class of G-protein coupled receptors (GPCR) that activate a variety of intracellular signaling pathways. Although features of P2Y receptor signaling in some cell types are known, the physiological roles of P2Y receptors in the nervous system are not well-characterized. In central, peripheral and sensory nervous systems, P2Y receptor activation profoundly affect glia, a cell type that plays important roles in nervous system development, function, and survival. Previous work has suggested a role for P2Y receptors in neurotransmission, neuronal-to-glial cell-cell signaling, alterations of gene expression, neuritogenesis, and interactions with growth factors in an additive or synergistic manner (Abbracchio and Burnstock, Jpn J Pharmacol, 78:113-45, 1998).
Hemostasis is the spontaneous process of stopping bleeding from damaged blood vessels. Precapillary vessels contract immediately when cut; within seconds, thrombocytes, or blood platelets, are bound to the exposed matrix of the injured vessel by a process called platelet adhesion. Platelets also stick to each other in a phenomenon known as platelet aggregation to form a platelet plug to stop bleeding quickly.
An intravascular thrombus results from a pathological disturbance of hemostasis. Platelet adhesion and aggregation are critical events in intravascular thrombosis. Activated under conditions of turbulent blood flow in diseased vessels or by the release of mediators from other circulating cells and damaged endothelial cells lining the vessel, platelets accumulate at a site of vessel injury and recruit further platelets into the developing thrombus. The thrombus can grow to sufficient size to block off arterial blood vessels. Thrombi can also form in areas of stasis or slow blood flow in veins. Venous thrombi can easily detach portions of themselves called emboli that travel through the circulatory system and can result in blockade of other vessels, such as pulmonary arteries. Thus, arterial thrombi cause serious disease by local blockade, whereas venous thrombi do so primarily by distant blockade, or embolization. These conditions include venous thrombosis, thrombophlebitis, arterial embolism, coronary and cerebral arterial thrombosis, unstable angina, myocardial infarction, stroke, cerebral embolism, kidney embolisms and pulmonary embolisms.
There is an unmet medical need for new therapeutic nucleotides that have good storage stability and/or in vivo stability that can be used for treating epithelial diseases, or for treating diseases or disorders associated with platelet aggregation with minimal side effects. Nucleotides, defined here as a nucleoside base with one or more phosphate groups attached at the furanosyl primary hydroxyl group, can act via receptors (e.g. P2Y), and ion channels (e.g. P2X). The therapeutic utility of nucleotides arises from their actions as either agonists or antagonists of receptor (P2) function. Two classes of therapeutic nucleotides have emerged recently-mononucleotides (e.g. nucleoside mono-, di-, and tri-phosphates) and dinucleotides (dinucleoside polyphosphates). Mononucleotides, such as uridine triphosphate and adenosine triphosphate (UTP and ATP) are potent ligands of P2 receptors (see U.S. Pat. Nos. 5,292,498 and 5,628,984). However these mononucleotides have poor chemical and metabolic stability making them less attractive as drug candidates due to required refrigeration and short in vivo half-life. Dinucleotides, such as diuridine tetraphosphate and diadenosine tretraphosphate (Up4U and Ap4A), show an improvement in chemical and metabolic stability (Yerxa, et al. (Drugs of the Future, 24:759-769 (1999)), while retaining activity at various P2 receptors (see U.S. Pat. Nos. 5,635,160; 5,837,861; 5,900,407; 6,319,908; and 6,323,187).
Despite the therapeutic improvements made by the use of dinucleotides and their in vivo and storage stability, the difficulty and expense of their synthesis requires further investigation of new class of compounds.